Background Optimization of the interventricular delay (VV-optimization) in cardiac resynchronization therapy (CRT) patients can be performed by evaluation of mechanical dyssynchrony. However, there is no consensus on which method to use. In this study, three conceptually different methods were evaluated. Methods Thirty consecutive CRT patients were included. At day 1, patients were atrioventricular and VV optimized by left ventricular outflow tract (LVOT) velocity time integral (VTI). At 6 months, 2D strain (2DS) echocardiography and tissue Doppler imaging (TDI) was performed at six different VV-programming delay in steps of 20 ms. LVOT and three indices of dyssynchrony were evaluated at each setting: standard deviation (SD) of time-to-peak strain in 12 segments (2DS-SD), SD of time-to-peak velocities in 12 segments (TDI-SD), and maximal activation delay (AD-max) by cross-correlation analysis (XCA) of TDI-derived myocardial acceleration curves. Results Feasibility was 90% for 2DS-SD and TDI-SD and 97% for AD-max. Coefficients of variation for intraobserver variability were 13% for 2DS-SD, 11% for TDI-SD, and 6% for AD-max. A relative increase in LVOT VTI > 10% was observed in 5/12 (42%) nonresponders and 7/18 (39%) responders to CRT. Optimization by all three dyssynchrony indices significantly increased LVOT VTI compared to simultaneous pacing and optimal setting at day 1 (P < 0.05, all). LVOT VTI was highest when using AD-max, and AD-max showed the best agreement (k = 0.71). Conclusion VV optimization at 6 months acutely benefits both responders and nonresponders; however, dyssynchrony indices do not perform equally well. XCA has a high feasibility and reproducibility and appears to be superior to time-to-peak techniques.